The objective of the present proposal is to gain basic information on the sequence and mechanism of the respiratory chain-linked energy transduction and the structure function relationship of mitochondrial inner membrane by studying various types of the respiratory chain-linked electron and energy transfer reactions in a bovine cardiac submitochondrial system. The following approaches will be employed. (1) Isolation and purification of homogenous membrane preparations by means of density gradient centrifugation; and characterization with respect to both biochemical and biophysical parameters of these preparations. (2) Localization of the non-heme iron sulfur proteins, cytochrome b, and c1 in the membrane by means of resolution and reconstitution. (3) Application of extrinsic probes for the study of membrane conformation and/or state changes. Continued effort on the environmental sensitive probes, ANS for hydrophobicity and quinacrine for local H ion will be made. Derivatives of quinacrine have been synthesized and will also be investigated. (4) Continued effort will be made on developing techniques to achieve highly coupled submitochondrial membrane preparations. This type of attack should expose the inadequacies of the existing hypotheses and theories, lead to new theories, clarify the delicate organization of the mitochondrial energy transfer system and ultimately lead to the understanding of the regulation of the mitochondrial energy metabolism in both healthy and diseased muscle.